Field of the Invention
The present invention relates to pullulanase chimeras, polynucleotides encoding the pullulanase chimeras, methods of producing the chimeras, and methods of using the chimeras. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides and to compositions comprising the pullulanases.
Description of the Related Art
Starch usually consists of about 80% amylopectin and 20% amylose. Amylopectin is a branched polysaccharide in which linear chains alpha-1,4 D-glucose residues are joined by alpha-1,6 glucosidic linkages. Amylopectin is partially degraded by alpha-amylase, which hydrolyzes the 1,4-alpha-glucosidic linkages to produce branched and linear oligosaccharides. Prolonged degradation of amylopectin by alpha-amylase results in the formation of so-called alpha-limit dextrins that are not susceptible to further hydrolysis by the alpha-amylase. Branched oligosaccharides can be hydrolyzed into linear oligosaccharides by a debranching enzyme. The remaining branched oligosaccharides can be depolymerized to D-glucose by glucoamylase, which hydrolyzes linear oligosaccharides into D-glucose.
Debranching enzymes which can attack amylopectin are divided into two classes: isoamylases (E.C. 3.2.1.68) and pullulanases (E.C. 3.2.1.41), respectively. Isoamylase hydrolyses alpha-1,6-D-glucosidic branch linkages in amylopectin and beta-limit dextrins and can be distinguished from pullulanases by the inability of isoamylase to attack pullulan, and by their limited action on alpha-limit dextrins.
It is well-known in the art to add isoamylases or pullulanases in starch conversion processes. Pullulanase is a starch debranching enzyme having pullulan 6-glucano-hydrolase activity (EC3.2.1.41) that catalyzes the hydrolyses the α-1,6-glycosidic bonds in pullulan, releasing maltotriose with reducing carbohydrate ends. Usually pullulanase is used in combination with an alpha amylase and/or a glucoamylase.
Pullulanases are known in the art. U.S. Pat. No. 6,074,854 and U.S. Pat. No. 5,817,498 disclose a pullulanase from Bacillus deramificans. WO2009/075682 discloses a pullulanase derived from Bacillus acidopullolyficus. 
The present invention provides polypeptides (pullulanase chimeras) having pullulanase activity and showing improved thermo-activity and/or thermo-stability compared to the parent pullulanases.